The Transcription Factor ThPOK Orchestrates Stochastic Interchromosomal Interactions Required for IFNB1 Virus-Inducible Gene Expression.

Virus infection induces stochastic activation of the interferon-ß gene. Three previously identified Alu-like DNA elements called NRCs (NF-κB reception centers) function by capturing and delivering NF-κB to the IFNB1 enhancer via stochastic interchromosomal interactions. We show that the transcription factor ThPOK binds cooperatively with NF-κB to NRCs and mediates their physical proximity with the IFNB1 gene via its ability to oligomerize when bound to DNA. ThPOK knockdown significantly decreased the frequency of interchromosomal interactions, NF-κB DNA binding to the IFNB1 enhancer, and virus-induced IFNB1 gene activation. We also demonstrate that cooperative DNA binding between ThPOK and NF-κB on the same face of the double DNA helix is required for interchromosomal interactions and distinguishes NRCs from various other Alu elements bearing κB sites. These studies show how DNA binding cooperativity of stereospecifically aligned transcription factors provides the necessary ultrasensitivity for the all-or-none stochastic cell responses to virus infection.

Time's up: bursting out of transcription.

Many inducible genes are transcribed in bursts. In this issue, Degenhardt et al. (2009) report computational models that predict and validate patterns of stochastic gene expression.

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs.

Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming.

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.

Stochastic phenotype switching leads to intratumor heterogeneity in human liver cancer.

Intratumor heterogeneity is increasingly recognized as a major factor impacting diagnosis and personalized treatment of cancer. We characterized stochastic phenotype switching as a mechanism contributing to intratumor heterogeneity and malignant potential of liver cancer. Clonal analysis of primary tumor cell cultures of a human sarcomatoid cholangiocarcinoma identified different types of self-propagating subclones characterized by stable (keratin-7-positive or keratin-7-negative) phenotypes and an unstable phenotype consisting of mixtures of keratin-7-positive and keratin-7-negative cells, which lack stem cell features but may reversibly switch their phenotypes. Transcriptome sequencing and immunohistochemical studies with the markers Zeb1 and CD146/MCAM demonstrated that switching between phenotypes is linked to changes in gene expression related but not identical to epithelial-mesenchymal transition. Stochastic phenotype switching occurred during mitosis and did not correlate with changes in DNA methylation. Xenotransplantation assays with different cellular subclones demonstrated increased tumorigenicity of cells showing phenotype switching, resulting in tumors morphologically resembling the invasive component of primary tumor and metastasis. CONCLUSION: Our data demonstrate that stochastic phenotype switching contributes to intratumor heterogeneity and that cells with a switching phenotype have increased malignant potential. (Hepatology 2017).

Retrograde signalling caused by heritable mitochondrial dysfunction is partially mediated by ANAC017 and improves plant performance.

Mitochondria are crucial for plant viability and are able to communicate information on their functional status to the cellular nucleus via retrograde signalling, thereby affecting gene expression. It is currently unclear if retrograde signalling in response to constitutive mitochondrial biogenesis defects is mediated by the same pathways as those triggered during acute mitochondrial dysfunction. Furthermore, it is unknown if retrograde signalling can effectively improve plant performance when mitochondrial function is constitutively impaired. Here we show that retrograde signalling in mutants defective in mitochondrial proteins RNA polymerase rpotmp or prohibitin atphb3 can be suppressed by knocking out the transcription factor ANAC017. Genome-wide RNA-seq expression analysis revealed that ANAC017 is almost solely responsible for the most dramatic transcriptional changes common to rpotmp and atphb3 mutants, regulating classical marker genes such as alternative oxidase 1a (AOX1a) and also previously-uncharacterised DUF295 genes that appear to be new retrograde markers. In contrast, ANAC017 does not regulate intra-mitochondrial gene expression or transcriptional changes unique to either rpotmp or atphb3 genotype, suggesting the existence of currently unknown signalling cascades. The data show that ANAC017 function extends beyond common retrograde transcriptional responses and affects downstream protein abundance and enzyme activity of alternative oxidase, as well as steady-state energy metabolism in atphb3 plants. Furthermore, detailed growth analysis revealed that ANAC017-dependent retrograde signalling provides benefits for growth and productivity in plants with mitochondrial defects. In conclusion, ANAC017 plays a key role in both biogenic and operational mitochondrial retrograde signalling, and improves plant performance when mitochondrial function is constitutively impaired.

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation.

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼ 5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

Quantifying the impact of wildfire smoke on solar photovoltaic generation in Australia.

The 2019-20 Australian wildfires caused extreme haze events across New South Wales (NSW), which reduced photovoltaic (PV) power output. We analyze 30-min energy data from 160 geographically separated residential PV systems in NSW with a total capacity of 312 kW from 6 Nov 2019-15 Jan 2020. The observed mean power reduction rate for PV energy generation as a function of the fine particulate matter (PM2.5) concentration is 13 ± 2% per 100 µg/m3 of PM2.5. The resulting energy loss for residential and utility PV systems is estimated at 175 ± 35 GWh, equating to a worst-case financial loss of 19 ± 4 million USD. We found the relative impact to be most significant in the mornings and evenings, which may necessitate the installation of additional energy storage. As PV systems are sensitive to smoke and become ubiquitous, we propose employing them to support wildfire detection and monitoring.